Background: In a previous article, we showed that metformin (MET) can reduce ionizing radiation (IR) induced apoptosis in human peripheral blood mononuclear cells. However, the anti-apoptotic mechanism of MET against IR remains unclear. The present study attempts to investigate the mechanism of action of MET in limiting X-ray induced apoptosis in human peripheral blood mononuclear cells. Material and Methods: Mononuclear cells were treated with MET for 2 hours and irradiated with 6 MV X-rays. The gene expression levels of BAX, CASP3 and BCL2 were determined 24 hours post irradiation using real time quantitative polymerase chain reaction (qPCR) technique. Furthermore, the protein levels of BAX, CASP3 and BCL2 were analyzed by Western blotting assay. Results: Radiation exposure increased the expressions of BAX and CASP3 genes, and decreased the expression of BCL2 gene in mononuclear cells. Conversely, an increase in BCL2 gene expression along with a decrease in BAX and CASP3 genes expression was observed in MET plus irradiated mononuclear cells. It was found that radiation increased BAX/BCL2 ratio, while MET pretreatment reduced these ratios. Also, treatment with MET without irradiation did not change the expressions of BAX, CASP3 and BCL2 genes. On the other hand, downregulated expression of BCL2 protein and upregulated expressions of BAX and CASP3 proteins were found in 2 Gy irradiated mononuclear cells, while pretreatment with MET significantly reversed this tendency. Conclusion: These results suggest that MET can protect mononuclear cells against apoptosis induced by IR through induction of cellular anti-apoptotic signaling.